X-chromosome inactivation (XCI) refers to the epigenetic silencing of one of the two active X chromosomes in female cells. XCI is essential for the normal development of placental female organisms. Females carrying only a single X chromosome suffer congenital cardiac and renal abnormalities and are generally infertile (Bondy 2009), while female organisms with two X chromosomes that do not undergo XCI die during early embryogenesis (Marahrens et al. 1997). These observations emphasize the necessity of maintaining an active as well as an inactive X chromosome (XaXi) in all somatic female cells throughout life and the importance of XCI for female mammalian development. Reprogramming of female somatic cells to pluripotency reactivates the somatically silent Xi and reestablishes the XaXa state typically found in ESCs (Navarro et al. 2008; Minkovsky et al. 2011). These data demonstrate an intimate link between X chromosome activity and pluripotency, yet it is unknown whether two active X chromosomes are necessary for the establishment and maintenance of the pluripotent state. The crucial molecule involved in initiation of XCI is the large non-coding RNA Xist, which is encoded on the X chromosome (Penny et al. 1996). Upon induction of differentiation in pluripotent cells, Xist RNA expression is exclusively upregulated on the future Xi. The RNA then spreads along the chromosome to initiate silencing in cis, via mechanisms that are still completely unclear. Prevailing models support the idea that the Xist RNA recruits silencing factors to the X chromosome to form an area of repressive chromatin (Wutz 2011). To determine the identity of these factors, we propose RNA-immunoprecipitation assays employing functional regions of the Xist RNA as bait in nuclear extracts derived from mouse ESCs (Aim 1). The identity of Xist RNA-associated proteins will be determined by mass spectrometric analysis. Mutant versions of the Xist RNA incapable of silencing will be used as controls. To determine the mechanisms of Xist RNA spreading, regions of the X chromosome bound by, or close to, the Xist RNA during the initial stages of XCI will be identified through chromatin immunoprecipitation experiments, again using the Xist RNA as bait (Aim 2). The co-immunoprecipitated chromatin fragments will be analyzed using high throughput DNA-sequencing and bioinformatics. Finally, to further understand the relationship between XCI and pluripotency, ectopic Xist RNA expression will be induced on one of the two X chromosomes in female mouse ESCs. The viability of these cells will be assessed using competition assays, while their potential to differentiate into all embryonic lineages will also be monitored by identification of differentiatin markers. The goals of these experiments are to generate new insights into the mechanisms of Xist RNA-dependent silencing during XCI and its relationship to maintenance of the stem cell state. As such, the initiation of XCI by Xist in differentiating ESCs provides an excellent model system with which to study the role of lncRNAs in epigenetic regulation and the establishment of cellular identity. PUBLIC HEALTH RELEVANCE: X-chromosome inactivation is an essential developmental process that occurs during early female embryogenesis and displays an intimate connection to the pluripotent state. It is a striking example of epigenetic regulation by a long non-coding RNA molecule, whose mechanisms of action can be readily studied ex vivo in embryonic stem cells. The work outlined in this proposal is aimed at understanding how a non-coding RNA molecule initiates extensive epigenetic changes that are critical to normal embryonic development and how these processes are related to the pluripotent state of the cell.